Chapter 4 —— 56 —— precision or fidelity can be ascertained by demonstrating that telomeric-sided and centromeric-sided junctions between exogenous and endogenous DNA sequences (jT and jC, respectively), result from HDR instead of imprecise end-joining processes (non-HDR). Hence, to assess the specificity and fidelity of DMD gene editing in human myoblasts co-transduced with AdVP.C9KARA and AAV-HRDMDGIN4, junction PCR screens were carried out on randomly selected mCherry-positive myoblast clones, where each clone represents an individual genome-modifying event. Of notice, in contrast to splice acceptor gene-trapping constructs, cell isolation based on constitutively active transgenes, such as the mCherry in AAV-HRDMD GIN4, avoids biased selection for on-target chromosomal DNA insertions. Hence, it is significant that strictly off-target chromosomal donor DNA insertions were not detected (i.e. jT-/jC-) in any of the randomly isolated myoblast lines and, amongst the forty lines analyzed, only two were not properly targeted as they lacked HDRderived chromosomal junctions at one of the two ends (i.e. jT+/jC-) (Supplementary Figure S11). Interestingly, similar to three myoblast lines that presented imprecise HDR-derived junctions, these two lines contained high molecular-weight products whose origins are consistent with AAV donor concatemers (Supplementary Figure S11). To test endogenous gene tagging via the dual viral vector genome-editing system at an independent locus, the AAV-HRLMNAGEX1 donor was assembled (Figure 4D). This AAV donor expresses gRNA GEX1 for site-specific DNA cleavage at exon 1 of LMNA and it contains a matched HR template for directional HDR-mediated fusion of the mScarlet-I reporter to the N-terminus of LMNA (Figure 4D). The LMNA protein is a component of the nuclear lamina matrix that locates underneath the inner nuclear membrane and whose roles include nuclear stability, chromatin structure and gene expression. Critically, LMNA mutations cause various disorders, e.g. Emery–Dreifuss muscular dystrophy, familial partial lipodystrophy, limb girdle muscular dystrophy, dilated cardiomyopathy and Hutchinson–Gilford progeria syndrome. Co-transduction experiments with AdVP.C9KARA and AAV-
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